Identification of two regulatory binding sites which confer myotube specific expression of the mono-ADP-ribosyltransferase ART1 gene.
BACKGROUND
Mono-ADP-ribosyltransferase (ART) 1 belongs to a family of mammalian ectoenzymes that catalyze the transfer of ADP-ribose from NAD+ to a target protein. ART1 is predominantly expressed in skeletal and cardiac muscle. It ADP-ribosylates alpha7-integrin which together with beta1-integrin forms a dimer and binds to laminin, a protein of the extracellular matrix involved in cell adhesion. This posttranslational modification leads to an increased laminin binding affinity.


RESULTS
Using C2C12 and C3H-10T 1/2 cells as models of myogenesis, we found that ART1 expression was restricted to myotube formation. We identified a fragment spanning the gene 1.3 kb upstream of the transcriptional start site as the functional promoter of the ART1 gene. This region contains an E box and an A/T-rich element, two conserved binding sites for transcription factors found in the promoters of most skeletal muscle specific genes. Mutating the DNA consensus sequence of either the E box or the A/T-rich element resulted in a nearly complete loss of ART1 promoter inducibility, indicating a cooperative role of the transcription factors binding to those sites. Gel mobility shift analyses carried out with nuclear extracts from C2C12 and C3H-10T 1/2 cells revealed binding of myogenin to the E box and MEF-2 to the A/T-rich element, the binding being restricted to C2C12 and C3H-10T 1/2 myotubes.


CONCLUSIONS
Here we describe the molecular mechanism underlying the regulation of the ART1 gene expression in skeletal muscle cells. The differentiation-dependent upregulation of ART1 mRNA is induced by the binding of myogenin to an E box and of MEF-2 to an A/T-rich element in the proximal promoter region of the ART1 gene. Thus the transcriptional regulation involves molecular mechanisms similar to those used to activate muscle-specific genes.
Friedrich
Maik
M
Institute of Biology II, Dept, of Immunobiology, University of Leipzig, Talstrasse 33, D-04103 Leipzig, Germany. Friedrich.Maik@gmx.net
Böhlig
Levin
L
Kirschner
Ralf D
RD
Engeland
Kurt
K
Hauschildt
Sunna
S
D036002:ADP Ribose Transferases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation; D002478:Cells, Cultured; D004285:Dogs; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D018008:Myogenin / Q000378:metabolism; D011401:Promoter Regions, Genetic; D050436:Regulatory Elements, Transcriptional / Q000235:genetics; D016415:Sequence Alignment
D018008:Myogenin; D036002:ADP Ribose Transferases